On April 25, 2009, Singapore implemented strict containment measures for pandemic (H1N1) 2009 with enhanced surveillance and hospital isolation. In the first month, seasonal influenza, predominantly virus subtype H3N2, was diagnosed for 32% of patients with acute febrile respiratory illness. Our findings underscore the high prevalence of seasonal influenza in Singapore.
Pandemic (H1N1) 2009; seasonal influenza; influenza; surveillance; viruses; Singapore; expedited; dispatch
Dengue re-emerges in Singapore despite decades of effective vector control; the infection predominantly afflicts adults. Severe dengue not fulfilling dengue hemorrhagic fever (DHF) criteria according to World Health Organization (WHO) 1997 guideline was increasingly reported. A new WHO 2009 guideline emphasized warning signs and a wider range of severe dengue manifestations. We aim to evaluate the utility of these two guidelines in confirmed adult dengue fatalities.
We conducted a multi-center retrospective chart review of all confirmed adult dengue deaths in Singapore from 1 January 2004 to 31 December 2008.
Of 28 adult dengue deaths, median age was 59 years. Male gender comprised 67.9% and co-morbidities existed in 75%. From illness onset, patients presented for admission at a median of 4 days and death occurred at a median of 12 days. Intensive care admission was required in 71.4%. Probable dengue was diagnosed in 32.1% by WHO 1997 criteria and 78.6% by WHO 2009. The earliest warning sign was persistent vomiting at a median of 1.5 days. Hematocrit change ≥20% concurrent with platelet count <20 × 10^9/L was associated with the shortest interval to death at a median of 3 days. Only 35.7% of death cases fulfilled DHF criteria by WHO 1997 versus severe dengue in 100.0% by WHO 2009 criteria. Deaths were due to shock and organ failure. Acute renal impairment occurred in 71.4%, impaired consciousness 57.1% and severe hepatitis 53.6%.
In our adult fatal dengue cohort, WHO 2009 criteria had higher sensitivity in diagnosing probable dengue and severe dengue compared with WHO 1997. As warning signs, persistent vomiting occurred early and hematocrit change ≥20% concurrent with platelet count <20 × 10^9/L preceded death most closely.
Only a minority of probable SARS cases caused transmission. We assess if any epidemiological or clinical factors in SARS index patients were associated with increased probability of transmission.
We used epidemiological and clinical data on probable SARS patients admitted to Tan Tock Seng Hospital. Using a case-control approach, index patients who had probable SARS who subsequently transmitted the disease to at least one other patient were analysed as "cases" against patients with no transmission as "controls", using multivariate logistic regression analysis.
98 index patients were available for analysis (22 with transmission, 76 with no transmission). Covariates positively associated with transmission in univariate analysis at p < 0.05 included delay to isolation (Day 7 of illness or later), admission to a non-isolation facility, pre-existing chronic respiratory disease and immunosuppressive disease, need for oxygen, shortness of breath, vomiting, and higher lactate dehydrogenase levels and higher neutrophil counts. In the multivariate analysis, only three factors were significant: delay to isolation, admission to a non-isolation facility and higher lactate dehydrogenase levels of >650 IU/L (OR 6.4, 23.8 and 4.7 respectively).
Clinical and epidemiological factors can help us to explain why transmission was observed in some instances but not in others.
A growing body of evidence suggests that dengue infection in Singapore predominantly occurs away from the home, but when and where dengue transmission occurs is unclear, confounding control efforts. The authors estimate days of the week in which dengue inpatients in Singapore were infected during the period 2006–2008, based on the day they became febrile and historical data on the incubation period, using Bayesian statistical methods. Among male inpatients, the relative risk of infection is an estimated 57% higher at the weekend, suggesting infections associated with the home or leisure activities. There was no evidence of elevated risk of infection at the weekend for female inpatients. The study motivates further research identifying locales frequented in the week leading up to onset to improve the effective targeting of vector control efforts.
HIV-1 subtype B and CRF01_AE are the predominant infecting subtypes among men who have sex with men (MSM) in Singapore. The genetic history, population dynamics and pattern of transmission networks of these genotypes remain largely unknown. We delineated the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and the recently characterized CRF51_01B strains circulating among the MSM population in Singapore. A total of 105 (49.5%) newly-diagnosed treatment-naïve MSM were recruited between February 2008 and August 2009. Phylogenetic reconstructions of the protease gene (HXB2: 2239 – 2629), gp120 (HXB2: 6942 – 7577) and gp41 (HXB2: 7803 – 8276) of the env gene uncovered five monophyletic transmission networks (two each within subtype B and CRF01_AE and one within CRF51_01B lineages) of different sizes (involving 3 – 23 MSM subjects, supported by posterior probability measure of 1.0). Bayesian coalescent analysis estimated that the emergence and dissemination of multiple sub-epidemic networks occurred between 1995 and 2005, driven largely by subtype B and later followed by CRF01_AE. Exponential increase in effective population size for both subtype B and CRF01_AE occurred between 2002 to 2007 and 2005 to 2007, respectively. Genealogical estimates suggested that the novel CRF51_01B lineages were probably generated through series of recombination events involving CRF01_AE and multiple subtype B ancestors. Our study provides the first insight on the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and CRF51_01B viral strains circulating among MSM in Singapore.
To identify demographic, clinical and laboratory risk factors for death due to dengue fever in adult patients in Singapore.
Multi-center retrospective study of hospitalized adult patients with confirmed dengue fever in Singapore between 1 January 2004 and 31 December 2008. Non-fatal controls were selected by matching age and year of infection with fatal cases. World Health Organization 1997, 2009 criteria were applied to define dengue hemorrhagic fever (DHF), warning signs and severe dengue. Statistical significance was assessed by conditional logistic regression modeling.
Significantly more fatal cases than matched controls had pre-existing co-morbid conditions, and presented with abdominal pain/tenderness. Median pulse rates were significantly higher while myalgia was significantly less frequent in cases. . Fatal cases also had higher leucocyte counts, platelet counts, serum sodium, potassium, urea, creatine and bilirubin levels on admission compared to controls. There was no statistical significant difference between the prevalence of DHF and hematocrit level among cases and controls. Multivariate analysis showed myalgia and leucocyte count at presentation were independent predictors of fatality (adjusted odds ratios 0.09 and 2.94 respectively). None of the controls was admitted to intensive care unit (ICU) or given blood transfusion, while 71.4% and 28.6% of fatal cases received ICU admission and blood transfusion.
Absence of myalgia and leucocytosis on admission were independently associated with fatality in our matched case-control study. Fatalities were also commonly associated with co-morbidities and clinicians should be alarmed if dengue patients fulfilled severe dengue case definition on admission.
Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4+ and CD8+ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4+ and 21 are CD8+ T cell epitopes. We observe that whereas CD8+ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4+ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4+ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4+ and CD8+ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.
MDR TB; transmission; Singapore; foreign-born; institutional; HIV/AIDS; correctional; viruses; tuberculosis and other mycobacteria; TB
Chikungunya virus (CHIKV) is an alphavirus which causes chronic and incapacitating arthralgia in humans. Although previous studies have shown that antibodies against the virus are produced during and after infection, the fine specificity of the antibody response against CHIKV is not known. Here, using plasma from patients at different times postinfection, we characterized the antibody response against various proteins of the virus. We have shown that the E2 and E3 glycoproteins and the capsid and nsP3 proteins are targets of the anti-CHIKV antibody response. Moreover, we have identified the different regions in these proteins which contain the linear epitopes recognized by the anti-CHIKV antibodies and determined their structural localization. Data also illustrated the effect of a single K252Q amino acid change at the E2 glycoprotein that was able to influence antibody binding and interaction between the antibodies and epitope because of the changes of epitope-antibody binding capacity. This study provides important knowledge that will not only aid in the understanding of the immune response to CHIKV infection but also provide new knowledge in the design of modern vaccine development. Furthermore, these pathogen-specific epitopes could be used for future seroepidemiological studies that will unravel the molecular mechanisms of human immunity and protection from CHIKV disease.
A recent HIV-1 molecular epidemiology survey in Singapore identified a novel CRF01_AE/B recombinant form, which accounted for 13 (11.9%) of 109 patient samples. Peripheral blood mononuclear cell DNA from three of these 13 patients was used to generate near full-length sequences to characterize the novel CRF01_AE/B recombinant form. The three isolates had a recombinant structure composed of CRF01_AE and subtype B, and shared identical breakpoints. As the three patients were not epidemiologically linked, this recombinant form has been designated CRF51_01B. Identification of the novel recombinant forms indicates ongoing active HIV-1 transmission in Singapore.
In 2009, the World Health Organization (WHO) proposed seven warning signs (WS) as criteria for hospitalization and predictors of severe dengue (SD). We assessed their performance for predicting dengue hemorrhagic fever (DHF) and SD in adult dengue.
DHF, WS and SD were defined according to the WHO 1997 and 2009 dengue guidelines. We analyzed the prevalence, sensitivity (Sn), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of WS before DHF and SD onset.
Of 1507 cases, median age was 35 years (5th–95th percentile, 17–60), illness duration on admission 4 days (5th–95th percentile, 2–6) and length of hospitalization 5 days (5th–95th percentile, 3–7). DHF occurred in 298 (19.5%) and SD in 248 (16.5%). Of these, WS occurred before DHF in 124 and SD in 65 at median of two days before DHF or SD. Three commonest warning signs were lethargy, abdominal pain/tenderness and mucosal bleeding. No single WS alone or combined had Sn >64% in predicting severe disease. Specificity was >90% for both DHF and SD with persistent vomiting, hepatomegaly, hematocrit rise and rapid platelet drop, clinical fluid accumulation, and any 3 or 4 WS. Any one of seven WS had 96% Sn but only 18% Sp for SD.
No WS was highly sensitive in predicting subsequent DHF or SD in our confirmed adult dengue cohort. Persistent vomiting, hepatomegaly, hematocrit rise and rapid platelet drop, and clinical fluid accumulation, as well as any 3 or 4 WS were highly specific for DHF or SD.
Dengue is a mosquito-borne infection with significant public health burden in tropical and subtropical regions. Clinical presentations may vary from self-limiting fever to severe dengue including death. The World Health Organization 2009 dengue guidelines classified dengue into dengue with and without warning signs, and severe dengue. In our adult dengue cohort, we found that lethargy, abdominal pain/tenderness, and mucosal bleeding occurred commonly. As predictors of severe dengue, these were not as specific as persistent vomiting, hepatomegaly, hematocrit rise and rapid platelet drop, and clinical fluid accumulation which occurred infrequently. Presence of any one warning sign had high sensitivity, but low specificity for severe dengue. Removal of lethargy from the list increased the specificity of any one warning sign, but sensitivity decreased. Consideration of these findings may avoid over hospitalization of potentially non-severe dengue patients and reduce burden on the healthcare system.
Chikungunya virus (CHIKV) is a mosquito-borne arthralgia arbovirus that is reemergent in sub-Saharan Africa and Southeast Asia. CHIKV infection has been shown to be self-limiting, but the molecular mechanisms of the innate immune response that control CHIKV replication remain undefined. Here, longitudinal transcriptional analyses of PBMCs from a cohort of CHIKV-infected patients revealed that type I IFNs controlled CHIKV infection via RSAD2 (which encodes viperin), an enigmatic multifunctional IFN-stimulated gene (ISG). Viperin was highly induced in monocytes, the major target cell of CHIKV in blood. Anti-CHIKV functions of viperin were dependent on its localization in the ER, and the N-terminal amphipathic α-helical domain was crucial for its antiviral activity in controlling CHIKV replication. Furthermore, mice lacking Rsad2 had higher viremia and severe joint inflammation compared with wild-type mice. Our data demonstrate that viperin is a critical antiviral host protein that controls CHIKV infection and provide a preclinical basis for the design of effective control strategies against CHIKV and other reemerging arthrogenic alphaviruses.
Whilst there have been studies on the risks and outcomes of MRSA colonization and infections in HIV-positive patients, local data is limited on the risk factors for MRSA colonization among these patients. We undertook this study in a tertiary HIV care centre to document the risk factors for colonization and to determine the prevalence of MRSA colonization among HIV-positive outpatients in Singapore.
This was a cross-sectional study in which factors associated with MRSA positivity among patients with HIV infection were evaluated. A set of standardized questionnaire and data collection forms were available to interview all recruited patients. Following the interview, trained nurses collected swabs from the anterior nares/axilla/groin (NAG), throat and peri-anal regions. Information on demographics, clinical history, laboratory results and hospitalization history were retrieved from medical records.
MRSA was detected in swab cultures from at least 1 site in 15 patients (5.1%). Inclusion of throat and/or peri-anal swabs increased the sensitivity of NAG screening by 20%. Predictors for MRSA colonization among HIV-positive patients were age, history of pneumonia, lymphoma, presence of a percutaneous device within the past 12 months, history of household members hospitalized more than two times within the past 12 months, and a most recent CD4 count less than 200.
This study highlights that a proportion of MRSA carriers would have been undetected without multiple-site screening cultures. This study could shed insight into identifying patients at risk of MRSA colonization upon hospital visit and this may suggest that a risk factor-based approach for MRSA surveillance focusing on high risk populations could be considered.
Methicillin-resistant Staphylococcus auerus; HIV; Risk factors; Colonization
We developed a probability equation and a decision tree from 1,973 predominantly dengue serotype 1 hospitalized adult dengue patients in 2004 to predict progression to dengue hemorrhagic fever (DHF), applied in our clinic since March 2007. The parameters predicting DHF were clinical bleeding, high serum urea, low serum protein, and low lymphocyte proportion. This study validated these in a predominantly dengue serotype 2 cohort in 2007. The 1,017 adult dengue patients admitted to Tan Tock Seng Hospital, Singapore had a median age of 35 years. Of 933 patients without DHF on admission, 131 progressed to DHF. The probability equation predicted DHF with a sensitivity (Sn) of 94%, specificity (Sp) 17%, positive predictive value (PPV) 16%, and negative predictive value (NPV) 94%. The decision tree predicted DHF with a Sn of 99%, Sp 12%, PPV 16%, and NPV 99%. Both tools performed well despite a switch in predominant dengue serotypes.
To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).
Since 2008, the Singapore Ministry of Health (MOH) has expanded HIV testing by increasing anonymous HIV test sites, as well as issuing a directive to hospitals to offer routine voluntary opt out inpatient HIV testing. We reviewed this program implemented at the end of 2008 at Tan Tock Seng Hospital (TTSH), the second largest acute care general hospital in Singapore.
Methods and Findings
From January 2009 to December 2010, all inpatients aged greater or equal than 21 years were screened for HIV unless they declined or were not eligible for screening. We reviewed the implementation of the Opt Out testing policy. There were a total of 93,211 admissions; 41,543 patients were included based on HIV screening program eligibility criteria. Among those included, 79% (n = 32,675) opted out of HIV screening. The overall acceptance rate was 21%. Majority of eligible patients who were tested (63%) were men. The mean age of tested patients was 52 years. The opt out rate was significantly higher among females (OR: 1.5, 95%CI: 1.4–1.6), aged >60 years (OR: 2.3, 95%CI: 2.2–2.4) and Chinese ethnicity (OR: 1.7, 95%CI:1.6–1.8). The false positive rate of the HIV screening test is 0.56%. The proportion of patients with HIV infection among those who underwent HIV screening is 0.18%. All16 confirmed HIV patients were linked to care.
The default opt-in rate of inpatient HIV testing was low at Tan Tock Seng Hospital, Singapore. Efforts to address individual HIV risk perception and campaigns against HIV stigma are needed to encourage more individuals to be tested for HIV.
Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. The immune mechanisms, which control viral multiplication and dissemination, are not yet known. Here, we studied the antibody response against the CHIKV surface antigens in infected patients. With plasma samples obtained during the early convalescent phase, we showed that the naturally-acquired IgG response is dominated by IgG3 antibodies specific mostly for a single linear epitope ‘E2EP3’. E2EP3 is located at the N-terminus of the E2 glycoprotein and prominently exposed on the viral envelope. E2EP3-specific antibodies are neutralizing and their removal from the plasma reduced the CHIKV-specific antibody titer by up to 80%. Screening of E2EP3 across different patient cohorts and in non-human primates demonstrated the value of this epitope as a good serology detection marker for CHIKV infection already at an early stage. Mice vaccinated by E2EP3 peptides were protected against CHIKV with reduced viremia and joint inflammation, providing a pre-clinical basis for the design of effective vaccine against arthralgia-inducing CHIKV and other alphaviruses.
CHIKV; linear neutralization epitopes; pre-clinical vaccine; protection; serology maker
Background. Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. Although infected individuals clear the virus from the blood, some develop debilitating and prolonged arthralgia.
Methods. We investigated specificity and strength of antibody responses in a longitudinal study on CHIKV-infected patients and analyzed their association with viral load, cytokine profile, and severity.
Results. We found that CHIKV-specific response is dominated by immunoglobulin G3 (IgG3) antibodies. The antibodies were neutralizing, and patients with high viremia rapidly developed high levels of anti-CHIKV antibodies of this specific isotype. Although these patients endured a more severe disease progression during the acute viremic phase, they cleared the virus faster and did not experience persistent arthralgia. However, significant persistent arthralgia was observed in patients with low viremia who developed IgG3 at a later stage.
Conclusions. Absence of early CHIKV-specific IgG3 may therefore serve as a specific marker of patients with increased risk of disease.
Background. Chikungunya virus (CHIKV) infection induces arthralgia. The involvement of inflammatory cytokines and chemokines has been suggested, but very little is known about their secretion profile in CHIKV-infected patients.
Methods. A case-control longitudinal study was performed that involved 30 adult patients with laboratory-confirmed Chikungunya fever. Their profiles of clinical disease, viral load, and immune mediators were investigated.
Results. When patients were segregated into high viral load and low viral load groups during the acute phase, those with high viremia had lymphopenia, lower levels of monocytes, neutrophilia, and signs of inflammation. The high viral load group was also characterized by a higher production of pro-inflammatory cytokines, such as interferon-α and interleukin (IL)–6, during the acute phase. As the disease progressed to the chronic phase, IL-17 became detectable. However, persistent arthralgia was associated with higher levels of IL-6 and granulocyte macrophage colony-stimulating factor, whereas patients who recovered fully had high levels of Eotaxin and hepatocyte growth factor.
Conclusions. The level of CHIKV viremia during the acute phase determined specific patterns of pro-inflammatory cytokines, which were associated with disease severity. At the chronic phase, levels of IL-6, and granulocyte macrophage colony-stimulating factor found to be associated with persistent arthralgia provide a possible explanation for the etiology of arthralgia that plagues numerous CHIKV-infected patients.
Influenza A virus; pandemics; seroepidemiologic study; cohort analysis; pandemic (H1N1) 2009; influenza; risk factors; travel; public transport; viruses; Singapore; adults; research
Estimation of influenza infection rates is important for determination of the extent of epidemic spread and for calculation of severity indicators. The authors compared estimated infection rates from paired and cross-sectional serologic surveys, rates of influenza like illness (ILI) obtained from sentinel general practitioners (GPs), and ILI samples that tested positive for influenza using data from similar periods collected during the 2009 H1N1 epidemic in Singapore. The authors performed sensitivity analyses to assess the robustness of estimates to input parameter uncertainties, and they determined sample sizes required for differing levels of precision. Estimates from paired seroconversion were 17% (95% Bayesian credible interval (BCI): 14, 20), higher than those from cross-sectional serology (12%, 95% BCI: 9, 17). Adjusted ILI estimates were 15% (95% BCI: 10, 25), and estimates computed from ILI and laboratory data were 12% (95% BCI: 8, 18). Serologic estimates were least sensitive to the risk of input parameter misspecification. ILI-based estimates were more sensitive to parameter misspecification, though this was lessened by incorporation of laboratory data. Obtaining a 5-percentage-point spread for the 95% confidence interval in infection rates would require more than 1,000 participants per serologic study, a sentinel network of 90 GPs, or 50 GPs when combined with laboratory samples. The various types of estimates will provide comparable findings if accurate input parameters can be obtained.
epidemics; estimation; infection; influenza, human; population surveillance; serologic tests; statistics as topic
Because healthcare facilities in many dengue endemic countries lack laboratory support, early dengue diagnosis must rely on either clinical recognition or a bedside diagnostic test. We evaluated the sensitivity and specificity of the 1997 and 2009 World Health Organization (WHO) dengue classification schemes and the NS1 strip test in acute sera from 154 virologically confirmed dengue patients and 200 patients with other febrile illnesses. Both WHO classification schemes had high sensitivity but lacked specificity. The NS1 strip test had high specificity, but its sensitivity was significantly lower in secondary compared with primary dengue infections. Differences in viral serotypes did not affect the performance of any of the three diagnostic approaches. Taken collectively, our findings indicate that the 1997 WHO dengue case definition can be used to exclude dengue, and the NS1 strip test can be used to confirm dengue infection, although the latter should be interpreted with caution in regions where secondary dengue infection is prevalent.
TOC Summary: Infection was associated with occupational and nonoccupational risk factors.
We describe incidence and risk factors for pandemic (H1N1) 2009 virus infection in healthcare personnel during the June–September 2009 epidemic in Singapore. Personnel contributed 3 serologic samples during June–October 2009, with seroconversion defined as a >4-fold increase in hemagglutination inhibition titers to pandemic (H1N1) 2009. Of 531 participants, 35 showed evidence of seroconversion. Seroconversion rates were highest in nurses (28/290) and lowest in allied health staff (2/116). Significant risk factors on multivariate analysis were being a nurse (adjusted odds ratio [aOR] 4.5, 95% confidence interval [CI] 1.0–19.6) and working in pandemic (H1N1) 2009 isolation wards (aOR 4.5, 95% CI 1.3–15.6). Contact with pandemic (H1N1) 2009–infected colleagues (aOR 2.5, 95% CI 0.9–6.6) and larger household size (aOR 1.2, 95% CI 1.0–1.4) were of borderline significance. Our study suggests that seroconversion was associated with occupational and nonoccupational risk factors.
Viruses; influenza; seroconversion; pandemic (H1N1) 2009; nosocomial infections; infection control; serology; vaccination; Singapore; research
An oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus evolved and emerged from zero to 52% of detectable virus within 48 hours of a patient’s exposure to oseltamivir. Phylogenetic analysis and data gathered by pyrosequencing and cloning directly on clinical samples suggest that the mutant emerged de novo.
Influenza; pandemic (H1N1) 2009; viruses; oseltamivir; antimicrobial resistance; Singapore; drug resistance; dispatch
The sensitivity and specificity of two in-house MAC-ELISA assays were tested and compared with the performance of commercially-available CTK lateral flow rapid test and EUROIMMUN IFA assays for the detection of anti-Chikungunya virus (CHIKV) IgM. Each MAC-ELISA assay used a whole virus-based antigen derived from genetically distinct CHIKV strains involved in two chikungunya disease outbreaks in Singapore (2008); a January outbreak strain with alanine at amino acid residue 226 of the E1 glycoprotein (CHIKV-A226) and a May-to-September outbreak strain that possessed valine at the same residue (CHIKV-226V). We report differences in IgM detection efficacy of different assays between the two outbreaks. The sensitivities of two PCR protocols were also tested.
Methods and Findings
For sera from January outbreak, the average detection threshold of CTK lateral flow test, MAC-ELISAs and EUROIMMUN IFA assays was 3.75, 4.38 and 4.88 days post fever onset respectively. In contrast, IgM detection using CTK lateral flow test was delayed to more than 7 days after fever onset in the second outbreak sera. However, MAC-ELISA using CHIKV-226V detected IgM in the second outbreak sera 3.96 days after fever onset, which was approximately one day earlier compared to the same assay using CHIKV-A226 (4.86 days). Specificity was 100% for both commercial assays, and 95.6% for the in-house MAC-ELISAs. For sensitivity determination of the PCR protocols, the probe-based real time RT-PCR method was found to be 10 times more sensitive than one based on SYBR Green.
Our findings suggested that the two strains of CHIKV using variants A226 and 226V resulted in variation in sensitivities of the assays evaluated. We postulated that the observed difference in antigen efficacy could be due to the amino acid substitution differences in viral E1 and E2 envelope proteins, especially the E1-A226V substitution. This evaluation demonstrates the importance of appraisal of different diagnostic assays before their application in clinical and operational settings.
Chikungunya is a mounting public health concern in many parts of the world. Definitive diagnosis is critical in differentiating the diseases, especially in dengue endemic areas. There are some commercial chikungunya kits and published molecular protocols available, but no comprehensive comparative evaluation of them was performed. Using sera collected in outbreaks caused by two variants of Chikungunya virus (A226 and 226V), we tested 2 commercial IgM tests (CTK lateral flow rapid test and EUROIMMUN IFA) alongside our in-house IgM assays (using both variants of the virus). Sensitivities of 2 published PCR protocols were also evaluated based on RNA standards derived from cell-cultured viruses. The commercial assays had different performances in each outbreak, with CTK's lateral flow test having the best performance in the first outbreak and EUROIMMUN IFA being more sensitive in the second outbreak. Use of the current circulating virus in a test assay improves sensitivity of the MAC-ELISAs. For PCR, a probe-based real time RT-PCR method was found to be 10 times more sensitive than the SYBR Green method. Despite this, the latter protocol is found to be more suitable and cost-effective for our diagnostic laboratory. This evaluation demonstrates the importance of appraisal of commercial kits and published protocols before application of a diagnostic tool in the clinical and operational setting.